P1527: NEXT GENERATION SEQUENCING IN HEREDITARY SPHEROCYTOSIS DIAGNOSIS
Authors: A. García Feria, J. Marco Buades, A. Zuñiga Cabrera, A. López Gabaldon, S. Broseta Tormos, M. I. Villa Velazquez, I. Fernandez Gayete, S. Gasco Clar, P. Rivera Garcia, E. Frances Aracil, E. Meseguer Martinez, O. Cortes Ortega, E. Donato Martinez, M. Fernandez Zarzoso, M. L. Juan Marco, A. Tolosa Muñoz, E. Gomez Beltran, M. J. Cejalvo Andujar, M. Panero Ruiz, P. Ribas Garcia, M. J. Fernandez Llavador, M. J. Sayas Lloris
Journal: HemaSphere (2022)

Background: Hereditary Spherocytosis (HS) is the most frequent cause of hereditary hemolytic anemia, clinically very heterogeneous. Diagnosis is made by family history, clinical history and presence of spherocytes in peripheral blood smear, but sometimes a combination of additional techniques is used for an adequate diagnosis, such EMA test, osmotic fragility test by flow cytometry (OFT), and recently, next generation sequencing (NGS) is being applied, which consists in the genetic study of the coding region and flanking intronic regions of the genes included in the TROMB1 V2 NGS panel, by the of ultrasequencing techniques. Aims: Show possible relationship between clinical behavior and NGS study in HS. Methods: Data has been collected from a total of 23 patients with a previous diagnosis of HS, of hematimetry tests such hemoglobin (Hb), mean corpuscular volumen (MCV), spherified MCV (SMCV), mean corpuscular hemoglobine (MCH), hemolysis (bilirubin, LDH, haptoglobin, reticulocytes), iron metabolism, % spherocytes, erythrocyte osmotic fragility (EOF), EMA test, OFT, and all these data have been compared together with possible patterns of anemia or hemolysis with NGS results. Ilumina (MiSeq) technology was used for sequencing. The Surelect custom gene panel with capture technology - Agilent Technologies- has been used. (genes panel is shown in table 1). Results: Results showed that all 23 patients had a pathological result on the NGS study. Among the multiple genes that code for erytrocyte membrane proteins, significant disease-related mutations were observed in four genes: SPTB (spectrin, beta), ANK1 (ankyrin 1), SLC4A1 (band 3/solute carrier family 4, member 1) and SPTA1 (spectrin, alpha 1). 16 significant mutations were found, of which 7 were previously known and 9 variants were novel mutations. The highest number of mutations was observed in SLC4A (n=10), followed by SPTB (n=6), ANK1 (n=6) and double heterozygosis for SPTB/SPTA1 (n=1). According to population databases (1000 genomes; ExAc; gnom AD), 11 mutations were pathological, 3 probably pathological and 2 of uncertain clinical significance. We observed that in the group of splenectomized patients (n=6) (26%)the most prevalent mutated gene was SLC4A1 (n=4), the group with slightly lower Hb concentration was ANK1 (Hb: 11.4 gr/dL and EOF 0.84%) and the pattern of hemolysis was higher in the SPTB gene group (reticulocytes 292.8 x103 mm3, total bilirubin 2.18 mg/dL, SMCV 63 fL and % spherocytes in peripheral blood: 9.6/field). The results of the genetic study were compared against OFT and EMA, these tests could be performed in all patients. OFT were negative in 4 patients (17.3%), of which 50% were in SLC41 group and 50% in SPTB group, being one of the mutations known with uncertain clinical degree and novel mutations in the other 3 patients, with pathological significance in one patient and with probably pathological repercussion in the other two. EMA tests were also negative in 4 patients (half of them also OFT negative, with one patient having a uncertainly significance in SLC4A gene and one patient in SPTB gene, a novel variant that was probably pathological). The other 2 patients had positive OFT, one patient with a known pathological variant in SLC4A and the other in ANK1 with an unknown mutation of uncertain significance. Summary/Conclusion: These results point out that there could be a relationship between the clinical diagnosis and the genetic profile of HS, according to the studied literature, and that NGS study can complement conventional tests to reach a more precise diagnosis of HS.